Carbohydrates are the most ubiquitous class of biomolecules in nature, yet glycan diversity imparts unique sugar features that can be targeted in a species-specific manner. The monosaccharide galactofuranose (Galf) is found in cell surface glycoconjugates of many human pathogens. However, Galf is absent from the mammalian glycome [Richards, M. R. and T. L. Lowary, Chemistry and Biology of Galactofuranose-Containing Polysaccharides. Chem Bio Chem, 2009. 10(12): p. 1920-1938], making enzymes involved in metabolism of the sugar attractive targets for treatment of human disease.
Galf contributes to virulence in infectious microbes such as Klebsiella pneumoniae (Kp) [Richards et al. 2009], Leishmania major, and Aspergillus fumigatus [Tefsen, B., et al., Galactofuranose in eukaryotes: aspects of biosynthesis and functional impact. Glycobiology, 2012. 22(4):456-469], as well as certain multicellular eukaryotic pathogens [Wesener, D. A., et al., UDP-galactopyranose mutase in nematodes. Biochemistry, 2013. 52(25):4391-8]. Mycobacterium tuberculosis, the causative agent of tuberculosis, harbors an essential Galf polysaccharide known as galactan within its renown thick and hydrophobic cell wall complex [Pan, F., et al., Cell Wall Core Galactofuran Synthesis Is Essential for Growth of Mycobacteria. J Bacteriol, 2001. 183(13):3991-3998]. Tuberculosis causes an estimated 2 million deaths worldwide every year (WHO report 2014). With the rise of multidrug-resistant and extremely drug resistant M. tuberculosis (Mt), tuberculosis is becoming increasingly difficult to treat (WHO report 2014). This underscores the need for new drug candidates in the pipeline.
Cell wall biosynthetic enzymes are targets of several first-line antitubercular drugs, including isoniazid and ethambutol [Richards et al. 2009; Pan et al. 2001]. Uridine 5′-diphosphate (UDP)-galactopyranose mutase (UGM) generates the biological source of UDP-Galf, utilized by galactofuranosyl transferases for construction of the mycobacterial cell wall galactan. UGM is a flavin-dependent protein that catalyzes ring contraction of UDP-galactopyranose (UDP-Galp) to form UDP-Galf [Soltero-Higgin, M., et al., A unique catalytic mechanism for UDP-galactopyranose mutase. Nat Struct Mol Biol, 2004. 11(6): 539-543] (FIG. 1), the five-membered ring isomer of nucleotide-linked galactose. UGM has been validated as a mycobacterial drug target [Dykhuizen, E. C., et al., Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth. J Am Chem Soc, 2008. 130(21):6706-7; Borrelli, S., et al., Antimycobacterial activity of UDP-galactopyranose mutase inhibitors. Int. J Antimicrob Agents, 2010. 36(4):364-8], and thus small molecule UGM antagonists are highly sought after.
Most efforts to develop UGM inhibitors have focused on UDP-sugar substrate analogs. (Caravano, A.; Dohi, H.; Sinay, P.; Vincent, S. P. Chem.-Eur. J. 2006, 12, 3114-3123; Liautard, V.; Christina, A. E.; Desvergnes, V.; Martin, O. R. J. Org. Chem. 2006, 71, 7337-7345; Ghavami, A.; Chen, J. J. W.; Pinto, B. M. Carbohydr. Res. 2004, 339, 401-407; Lee, R. E.; Smith, M. D.; Pickering, L.; Fleet, G. W. J. Tetrahedron Lett. 1999, 40:8689-8692; Liautard, V.; Desvergnes, V.; Martin, O. R. Org. Lett. 2006, 8, 1299-1302.) Simple sugar derivatives, including galactopyranose or galactofuranose analogs, bind weakly with affinities in the millimolar range (Lee, R. E.; Smith, M. D.; Nash, R. J.; Griffiths, R. C.; McNeil, M.; Grewal, R. K.; Yan, W. X.; Besra, G. S.; Brennan, P. J.; Fleet, G. W. J. Tetrahedron Lett. 1997, 38, 6733-6736; Veerapen, N.; Yuan, Y.; Sanders, D. A. R.; Pinto, B. M. Carbohydr. Res. 2004, 339, 2205-2217.) Inhibitors that incorporate the uridine portion of the substrate bind substantially better, with affinities that approximate that of UDP-Galp (Kd=52 μM) (Itoh, K.; Huang, Z. S.; Liu, H. W. Org. Lett. 2007, 9, 879-882; Caravano, A.; Vincent, S. P.; Sinay, P. Chem. Commun. 2004, 1216-1217; Caravano, A.; Mengin-Lecreulx, D.; Brondello, J. M.; Vincent, S. P.; Sinay, P. Chem.-Eur. J. 2003, 9, 5888-5898; Pan, W. D.; Ansiaux, C.; Vincent, S. P. Tetrahedron Lett. 2007, 48, 4353-4356; Scherman, M. S.; Winans, K. A.; Stern, R. J.; Jones, V.; Bertozzi, C. R.; McNeil, M. R. Antimicrob. Agents Chemother. 2003, 47, 378-382.) These approaches have not yet afforded compounds that block mycobacterial growth.
Certain non-substrate based molecules have been identified as UMG ligands. For example, certain nitrofuranylamides have been identified as inhibitors of UGM catalysis and mycobacterial growth. (Tangallapally, R. P.; Yendapally, R.; Lee, R. E.; Hevener, K.; Jones, V. C.; Lenaerts, A. J. M.; McNeil, M. R.; Wang, Y. H.; Franzblau, S.; Lee, R. E. J. Med. Chem. 2004, 47, 5276-5283.) Nevertheless, the UGM inhibition and antimycobacterial activity of these compounds were not correlated, so they do not address the utility of inhibiting UGM.
Published International application WO 2005/007625 (Lee et al.), as well as published U.S. application 20050222408, relate to certain heterocyclic amides with anti-tuberculosis activity. More specifically, these patent documents relate to compounds of formula:
wherein A is selected from the group consisting of oxygen, sulfur, and NR15, where R15 is selected from the group consisting of H, alkyl, aryl, substituted alkyl, and substituted aryl; B, D, and E are each independently selected from the group consisting of CH, nitrogen, sulfur and oxygen; R1 is selected from the group consisting of nitro, halo, alkyl ester, arylsulfanyl, arylsulfinyl, arylsulfonyl and sulfonic acid; t is an integer from 1 to 3; and X is a substituted amide. These patent documents are incorporated by reference herein at least in part for the definitions of structural elements of the above formula.
A fluorescence polarization (FP) based assay has been developed that allows detection of competitive UGM inhibitors [Soltero-Higgin, M., et al., Identification of inhibitors for UDP-galactopyranose mutase. J Am Chem Soc, 2004. 126(34): p. 10532-3; Dykhuizen, E. C. and L. L. Kiessling, Potent ligands for prokaryotic UDP-galactopyranose mutase that exploit an enzyme subsite. Org Lett, 2009. 11(1): p. 193-6]. Using this FP assay, several high-throughput screens (HTS) have been conducted, endeavoring discovery of small molecule UGM ligands [Soltero-Higgin et al. 2004; Carlson, E. E., J. F. May, and L. L. Kiessling, Chemical probes of UDP-galactopyranose mutase. Chem Biol, 2006. 13(8): p. 825-37]. The screens culminated in fairly low hit rates, revealing the challenging nature of UGM as a target. Nonetheless, through HTS, one series of thiazolidinones (TZ) with considerable activity towards UGM was discovered [Soltero-Higgin et al. 2004]. The TZ series was optimized through scaffold hopping to a 2-aminothiazole (AT) inhibitor core [Dykhuizen, et al., 2008]. The most potent AT displays an IC50 of 7.2 μM and 37 μM against KpUGM and MtUGM, respectively [Dykhuizen, et al. 2008; Borrelli et al. 2010].
U.S. Pat. No. 8,273,778 issued Sep. 25, 2012 relates to inhibitors of UDP-galactopyranose mutase having among others, 2-aminothiazole structures. This issued patent is incorporated by reference herein in its entirety for descriptions of the UDP-galactopyranose mutase inhibitors there as well as methods of assessing such inhibitors and methods of application of such inhibitors.
While a number of small molecule inhibitors of UDP-galactopyranose mutase have been identified, there remains a need in the art for additional inhibitors which exhibit effective inhibition of microorganisms having UDP-galactopyranose mutase.